The purpose of this revised renewal application is to further understand how DeltaFosB (DFosB) increases bone formation (BF) in adult mice. Unlike most other transcription factors involved in osteoblast (OB) differentiation, DFosB increases BF postnatally, at any point in time, while not affecting skeletal development. This mimics the desired effects of a true bone anabolic therapeutic agent. We have now shown that the further truncated D2DFosB isoform recapitulates the phenotype of DFosB in bone. Like Fra1 and DNJunD, which induce a similar phenotype, D2DFosB does not have intrinsic transcriptional activity and antagonizes AP1. D2DFosB's action may be primarily within the transcriptional machinery since all interactions we have identified are with transcription factors or co-factors (Juns, Runx2, C/EBPb, Smad6 and Zfp521, a novel key player in the regulation of BF). In contrast to FosB, D2DFosB fails to repress beta-catenin transactivation in response to Wnt3a or to induce the inhibitory Smad6 inresponse to BMP2. These are major breakthroughs in our understanding of the DFosB osteosclerotic phenotype: the increased BF when DfosB, and therefore D2DFosB, is expressed may result not only from its positive effects on BMP signaling and Runx2 but also from the loss of the negative effects that full length FosB exerts on BMP and Wnt signaling. We propose to further explore the mechanisms by which D2DFosB affects BF, focusing on the AP-1 machinery and BMP and Wnt signaling. The specific aims of this renewal are therefore: Aim 1: Further Explore the physiological roles of the FosB isoforms in vivo through analysis of; - A (FosB + (2(FosB "knockout" mouse, expressing only FosB, by knocking-in an unspliceable mutant form of FosB - A (FosB "knock-in" mouse, expressing only (FosB and (2(FosB, - A transgenic mouse overexpressing an unspliceable mutant of FosB in osteoblasts, overexpressing only FosB. Aim 2: Identify the FosB domains required to affect osteoblast differentiation and function; Aim 3: Characterize the role of FosB proteins and their domains and interactions in the BMP and Wnt signaling pathways. The experiments proposed in this application could therefore lead to the identification of novel pathways regulating bone formation and novel targets for drug discovery, potentially allowing new approaches for anabolic therapeutic intervention in osteoporosis, osteogenesis imperfecta and other diseases where bone mass is decreased. PUBLIC HEALTH RELEVANCE: The purpose of this application is to further understand the molecular mechanisms by which a transcription factor called (FosB increases bone density in adult mice. Unlike most other transcription factors involved in the differentiation of osteoblasts (bone- forming cells), (FosB increases bone formation postnatally, at any point in time, while not affecting skeletal development, mimicking the desired effects of a true bone anabolic therapeutic agent. The experiments proposed in this application could therefore lead to the identification of novel pathways regulating bone formation and novel targets for drug discovery, potentially allowing new approaches for anabolic therapeutic intervention in osteoporosis, osteogenesis imperfecta and other diseases where bone mass is decreased.